MHC class II molecules present peptides derived from self and foreign antigens to CD4+ T-cells. Peptides derived from exogenous antigens and proteins localized in endosomes, are captured by class II dimers as these MHC molecules transit to the cell surface. Remarkably, epitopes from select cytoplasmic antigens also gain access to class II proteins. Cytoplasmic antigens have long been viewed as the primary source of peptides for MHC class molecules. Yet mechanisms appear to have evolved to allow class II proteins to sample select peptides within the cytoplasm, potentiating helper T-cell responses to intracellular pathogens, tumors and the maintenance of self tolerance. The purpose of this application is to elucidate the pathway(s) required for processing and delivery of cytoplasmic antigens to MHC class II proteins. We hypothesized that proteases in the cytoplasm regulate epitope abundance, with a subset of the resulting peptides being directly translocated into membrane organelles for binding to class II molecules. Indeed, neutral proteases within the cytoplasm can modulate epitope production for class II restricted presentation. In aim 1, studies are proposed to further identify these proteases and to define their regulation in APC. Class II-restricted presentation of exogenous antigens is controlled by co-factors such as the Invariant chain, DM and DO. In aim 2, the importance of these accessory molecules in cytoplasmic antigen presentation will be examined. As these co-factors associate with class II proteins in distinct membrane organelles, elucidating their role in cytoplasmic antigen presentation will help dissect the relative importance of the endoplasmic reticulum vs. endosomes in this novel class II pathway. Studies also suggest that translocation of cytoplasmic epitopes into distinct membrane organelles must occur to deliver these ligands to class II molecules. Yet, the essential features guiding cytoplasmic epitope translocation to receptive class II proteins, remain undefined. In aim 3, experiments to elucidate the role of peptide transporters and heat shock proteins in cytoplasmic antigen presentation via MHC class II molecules are therefore proposed. These studies will provide key insights into this novel pathway for class II presentation with implications for vaccine development.